1. Neoplasia Lecture 4 Dr. Maha Arafah Dr. Abdulmalik Alsheikh, MD, FRCPC CARCINOGENESIS Foundation block 2014 Pathology

2. CARCINOGENESIS Carcinogenesis is a multistep process at both the phenotypic and the genetic levels. Carcinogenesis is a multistep process at both the phenotypic and the genetic levels. It starts with a genetic damage: It starts with a genetic damage: Environmental Environmental Chemical Chemical Radiation Radiation Viral Viral Inhereted Inhereted

3. Carcinogenesis Genetic damage lead to “ mutation” Genetic damage lead to “ mutation” single cell which has the genetic damage undergoes neoplastic proliferation ( clonal expansion) forming the tumor mass single cell which has the genetic damage undergoes neoplastic proliferation ( clonal expansion) forming the tumor mass

5. Carcinogenesis Where are the targets of the genetic damage? Where are the targets of the genetic damage?

6. 1.Growth promoting protooncogenes 1.Protooncogene > mutation > oncogene 2.Growth inhibiting (supressors) genes 3.Genes regulating apoptosis 4.DNA repair genes

7. Carcinogenesis Main changes in the cell physiology that lead to formation of the malignant phenotype: Main changes in the cell physiology that lead to formation of the malignant phenotype: Self-sufficiency in growth signals Self-sufficiency in growth signals Insensitivity to growth-inhibitory Insensitivity to growth-inhibitory signals signals Evasion of apoptosis Evasion of apoptosis Limitless replicative potential Limitless replicative potential Sustained angiogenesis Sustained angiogenesis Ability to invade and metastsize Ability to invade and metastsize

8. Carcinogenesis A - Self-sufficiency in Growth signals: Oncogene: Gene that promote autonomous cell growth in cancer cells Oncogene: Gene that promote autonomous cell growth in cancer cells They are derived by mutations in protooncogenes They are derived by mutations in protooncogenes They are characterized by the ability to promote cell growth in the absence of normal growth- promoting signals They are characterized by the ability to promote cell growth in the absence of normal growth- promoting signals Oncoproteins : are the products Oncoproteins : are the products

9. Carcinogenesis Remember the cell cycle Remember the cell cycle Binding of a growth factor to its receptor on the cell membrane Binding of a growth factor to its receptor on the cell membrane Activation of the growth factor receptor leading to activation of signal-transducing proteins Activation of the growth factor receptor leading to activation of signal-transducing proteins Transmission of the signal to the nucleus Transmission of the signal to the nucleus Induction of the DNA transcription Induction of the DNA transcription Entry in the cell cycle and cell division Entry in the cell cycle and cell division

10. Carcinogenesis HOW CANCER CELLS ACQUIRE SELF- SUFFICIENCY IN GROWTH SIGNALS?? HOW CANCER CELLS ACQUIRE SELF- SUFFICIENCY IN GROWTH SIGNALS??

11. Carcinogenesis 1- Growth factors: Cancer cells are capable to synthesize the same growth factors to which they are responsive E.g. Sarcomas ---- > TGF-  Glioblastoma-----> PDGF

12. Carcinogenesis 2-Growth factors receptors: Receptors --- mutation ----continous signals to cells and uncontroled growth Receptors --- mutation ----continous signals to cells and uncontroled growth Receptors --- overexpression ---cells become very sensitive ----hyperresponsive to normal levels of growth factors Receptors --- overexpression ---cells become very sensitive ----hyperresponsive to normal levels of growth factors

14. Carcinogenesis Example : Example : Epidermal Growth Factor ( EGF ) Receptor family Epidermal Growth Factor ( EGF ) Receptor family HER2 HER2 Amplified in breast cancers and other tumors Amplified in breast cancers and other tumors High levels of HER2 in breast cancer indicate poor prognosis High levels of HER2 in breast cancer indicate poor prognosis Anti- HER2 antibodies are used in treatment Anti- HER2 antibodies are used in treatment

15. Carcinogenesis 3- Signal-transducing proteins : They receive signals from activated growth factors receptors and transmitte them to the nucleus. Examples : They receive signals from activated growth factors receptors and transmitte them to the nucleus. Examples : RAS RAS ABL ABL

16. Carcinogenesis RAS : RAS : 30% of all human tumors contain mutated RAS gene. E.g : colon. Pancreas cancers 30% of all human tumors contain mutated RAS gene. E.g : colon. Pancreas cancers Mutations of the RAS gene is the most common oncogene abnormality in human tumors Mutations of the RAS gene is the most common oncogene abnormality in human tumors Mutations in RAS --- cells continue to proliferate Mutations in RAS --- cells continue to proliferate

18. Carcinogenesis ABL gene ABL gene ABL protooncogene has a tyrosine kinase activity ABL protooncogene has a tyrosine kinase activity Its activity is controlled by negative regulatory mechanism Its activity is controlled by negative regulatory mechanism E.g. : chronic myeloid leukemia ( CML ) : E.g. : chronic myeloid leukemia ( CML ) : t( 9,22) ---ABL gene transferred from ch. 9 to ch. 22 t( 9,22) ---ABL gene transferred from ch. 9 to ch. 22 Fusion with BCR ---> BCR-ABL Fusion with BCR ---> BCR-ABL BCR-ABL has tyrosine kinase acttivity ---( oncogene) BCR-ABL has tyrosine kinase acttivity ---( oncogene)

19. CML patients are treated with ( Gleevec) which is inhibitor of kinase

20. Carcinogenesis 4- Nuclear transcription factors : Mutations may affect genes that regulate transcription of DNA  growth autonomy Mutations may affect genes that regulate transcription of DNA  growth autonomy E.g. MYC E.g. MYC MYC protooncogene produce MYC protein when cell receives growth signals MYC protooncogene produce MYC protein when cell receives growth signals MYC protein binds to DNA leading to activation of growth-related genes MYC protein binds to DNA leading to activation of growth-related genes

21. Carcinogenesis Normally … MYC decrease when cell cycle begins …but..in tumors there is sustained expression of MYC  continuous proliferation Normally … MYC decrease when cell cycle begins …but..in tumors there is sustained expression of MYC  continuous proliferation E.g. Burkitt Lymphoma ; MYC is dysregulated due to t( 8,14) E.g. Burkitt Lymphoma ; MYC is dysregulated due to t( 8,14)

22. Carcinogenesis 5- Cyclins and cyclins- dependent kinases (CDKs) Progression of cells through cell cycles is regulated by CDKs after they are activated by binding with cyclins Progression of cells through cell cycles is regulated by CDKs after they are activated by binding with cyclins Mutations that dysregulate cyclins and CDKs will lead to cell proliferation …e.g. Mutations that dysregulate cyclins and CDKs will lead to cell proliferation …e.g. Cyclin D genes are overexpressed in breast, esophagus and liver cancers. Cyclin D genes are overexpressed in breast, esophagus and liver cancers. CDK4 is amplified in melanoma and sarcomas CDK4 is amplified in melanoma and sarcomas

24. Carcinogenesis Main changes in the cell physiology that lead to formation of the malignant phenotype: Main changes in the cell physiology that lead to formation of the malignant phenotype: A- Self-sufficiency in growth signals B- Insensitivity to growth-inhibitory signals C- Evasion of apoptosis D- Limitless replicative potential E- Sustained angiogenesis F- Ability to invade and metastsize

25. Carcinogenesis 2. Insensitivity to growth-inhibitory signals Tumor suppressor genes control ( apply brakes) cells proliferation Tumor suppressor genes control ( apply brakes) cells proliferation If mutation caused disruption to them  cell becomes insensitive to growth inhibition  uncontrolled proliferation If mutation caused disruption to them  cell becomes insensitive to growth inhibition  uncontrolled proliferation Examples: RB, TGF- , APC, P53 Examples: RB, TGF- , APC, P53

27. Carcinogenesis RB ( retinoblastoma ) gene : RB ( retinoblastoma ) gene : First tumor supressor gene discovered First tumor supressor gene discovered It was discovered initially in retinoblastomas It was discovered initially in retinoblastomas Found in other tumors, e.g. breast ca Found in other tumors, e.g. breast ca RB gene is a DNA-binding protein RB gene is a DNA-binding protein RB is located on chromosome 13 RB is located on chromosome 13

28. Carcinogenesis RB gene exists in “ active “ and “ inactive” forms RB gene exists in “ active “ and “ inactive” forms If active  will stop the advancing from G1 to S phase in cell cycle If active  will stop the advancing from G1 to S phase in cell cycle If cell is stimulated by growth factors  inactivation of RB gene  brake is released  cells start cell cycle …G1  S  M …then RB gene is activated again If cell is stimulated by growth factors  inactivation of RB gene  brake is released  cells start cell cycle …G1  S  M …then RB gene is activated again

30. Carcinogenesis Retinoblastoma is an uncommon childhood tumor Retinoblastoma is an uncommon childhood tumor Retinoblastoma is either sporadic (60%) or familial ( 40% ) Retinoblastoma is either sporadic (60%) or familial ( 40% ) Two mutations required to produce retinoblastoma Two mutations required to produce retinoblastoma Both normal copies of the gene should be lost to produce retinoblastoma Both normal copies of the gene should be lost to produce retinoblastoma

32. Carcinogenesis Transforming Growth Factor-  pathway: Transforming Growth Factor-  pathway: TGF-  is an inhibitor of proliferation TGF-  is an inhibitor of proliferation It regulate RB pathway It regulate RB pathway Inactivation of TGF-  lead to cell proliferation Inactivation of TGF-  lead to cell proliferation Mutations in TGF-  pathway are present in :  of pancreatic cancers  of colon cancers

33. Carcinogenesis Adenomatous Polyposis Coli –  Catenin pathway: Adenomatous Polyposis Coli –  Catenin pathway: APC is tumor supressor gene APC is tumor supressor gene APC gene loss is very common in colon cancers APC gene loss is very common in colon cancers It has anti-proliferative action through inhibition of  Catenin which activate cell proliferation It has anti-proliferative action through inhibition of  Catenin which activate cell proliferation Individuals with mutant APC develop thousands of colonic polyps Individuals with mutant APC develop thousands of colonic polyps

34. Adenomatous Polyposis Coli

35. Carcinogenesis One or more of the polyps will progress to colonic carcinoma One or more of the polyps will progress to colonic carcinoma APC mutations are seen in 70% to 80% of sporadic colon cancers APC mutations are seen in 70% to 80% of sporadic colon cancers

36. Carcinogenesis P53 P53 It has multiple functions It has multiple functions Mainly : Mainly : Tumor suppressor gene ( anti-proliferative ) Tumor suppressor gene ( anti-proliferative ) Regulates apoptosis Regulates apoptosis

37. Carcinogenesis P53 senses DNA damage P53 senses DNA damage Causes G1 arrest to give chance for DNA repair Causes G1 arrest to give chance for DNA repair Induce DNA repair genes Induce DNA repair genes If a cell with damaged DNA cannot be repaired, it will be directed by P53 to undergo apoptosis If a cell with damaged DNA cannot be repaired, it will be directed by P53 to undergo apoptosis

38. Carcinogenesis With loss of P53, DNA damage goes unrepaired With loss of P53, DNA damage goes unrepaired Mutations will be fixed in the dividing cells, leading to malignant transformation Mutations will be fixed in the dividing cells, leading to malignant transformation

40. P53 is called the “ guardian of the genome” P53 is called the “ guardian of the genome” 70% of human cancers have a defect in P53 70% of human cancers have a defect in P53 It has been reported with almost all types of cancers : e.g. lung, colon, breast It has been reported with almost all types of cancers : e.g. lung, colon, breast In most cases, mutations are acquired, but can be inhereted, e.g : Li-Fraumeni syndrome In most cases, mutations are acquired, but can be inhereted, e.g : Li-Fraumeni syndrome Carcinogenesis

41. Carcinogenesis Main changes in the cell physiology that lead to formation of the malignant phenotype: Main changes in the cell physiology that lead to formation of the malignant phenotype: A- Self-sufficiency in growth signals B- Insensitivity to growth-inhibitory signals C- Evasion of apoptosis D- Limitless replicative potential E- Sustained angiogenesis F- Ability to invade and metastsize

42. Carcinogenesis Evasion of apoptosis: Evasion of apoptosis: Mutations in the genes regulating apoptosis are factors in malignant transformation Mutations in the genes regulating apoptosis are factors in malignant transformation Cell survival is controlled by genes that promote and inhibit apoptosis Cell survival is controlled by genes that promote and inhibit apoptosis

43. Evasion of apoptosis Evasion of apoptosis Reduced CD95 level inactivate death – induced signaling cascade that cleaves DNA to cause death  tumor cells are less susceptible to apoptosis Reduced CD95 level inactivate death – induced signaling cascade that cleaves DNA to cause death  tumor cells are less susceptible to apoptosis DNA damage induced apoptosis (with the action of P53 ) can be blocked in tumors DNA damage induced apoptosis (with the action of P53 ) can be blocked in tumors loss of P53 and up- regulation of BCL2 prevent apoptosis e.g. follicular lymphoma loss of P53 and up- regulation of BCL2 prevent apoptosis e.g. follicular lymphoma

44. Carcinogenesis Main changes in the cell physiology that lead to formation of the malignant phenotype: Main changes in the cell physiology that lead to formation of the malignant phenotype: A- Self-sufficiency in growth signals B- Insensitivity to growth-inhibitory signals C- Evasion of apoptosis D- Limitless replicative potential E- Sustained angiogenesis F- Ability to invade and metastsize

45. Limitless replicative potential: Limitless replicative potential: Normally there is progressive shortening of telomeres at the ends of chromosomes Normally there is progressive shortening of telomeres at the ends of chromosomes Telomerase is active in normal stem cells but absent in somatic cells Telomerase is active in normal stem cells but absent in somatic cells In tumor cells : activation of the enzyme telomerase, which can maintain normal telomere length In tumor cells : activation of the enzyme telomerase, which can maintain normal telomere length

46. Carcinogenesis Main changes in the cell physiology that lead to formation of the malignant phenotype: Main changes in the cell physiology that lead to formation of the malignant phenotype: A- Self-sufficiency in growth signals B- Insensitivity to growth-inhibitory signals C- Evasion of apoptosis D- Limitless replicative potential E- Sustained angiogenesis F- Ability to invade and metastsize

47. Carcinogenesis Sustained angiogenesis Sustained angiogenesis Neovascularization has two main effects: Neovascularization has two main effects: Perfusion supplies oxygen and nutrients Perfusion supplies oxygen and nutrients Newly formed endothelial cells stimulate the growth of adjacent tumor cells by secreting growth factors, e.g : PDGF, IL-1 Newly formed endothelial cells stimulate the growth of adjacent tumor cells by secreting growth factors, e.g : PDGF, IL-1 Angiogenesis is required for metastasis Angiogenesis is required for metastasis

48. How do tumors develop a blood supply? How do tumors develop a blood supply? Tumor-associated angiogenic factors Tumor-associated angiogenic factors These factors may be produced by tumor cells or by inflammatory cells infiltrating the tumor e.g. macrophages These factors may be produced by tumor cells or by inflammatory cells infiltrating the tumor e.g. macrophages Important factors : Important factors : Vascular endothelial growth factor( VEGF ) Vascular endothelial growth factor( VEGF ) Fibroblast growth factor Fibroblast growth factor

50. Carcinogenesis Main changes in the cell physiology that lead to formation of the malignant phenotype: Main changes in the cell physiology that lead to formation of the malignant phenotype: A- Self-sufficiency in growth signals B- Insensitivity to growth-inhibitory signals C- Evasion of apoptosis D- Limitless replicative potential E- Sustained angiogenesis F- Ability to invade and metastsize

52. Carcinogenesis Ability to invade and metastsize: Ability to invade and metastsize: Two phases : Two phases : Invasion of extracellular matrix Invasion of extracellular matrix Vascular dissimenation and homing of tumor cells Vascular dissimenation and homing of tumor cells

53. Carcinogenesis Invasion of ECM: Invasion of ECM: Malignant cells first breach the underlying basement membrane Malignant cells first breach the underlying basement membrane Traverse the interstitial tissue Traverse the interstitial tissue Penetrate the vascular basement membrane Penetrate the vascular basement membrane Gain access to the circulation Gain access to the circulation  Invasion of the ECM has four steps:

54. 1. Detachment of tumor cells from each other

55. 2. Attachments of tumor cells to matrix components

56. 3. Degradation of ECM by collagenase enzyme

57. 4. Migration of tumor cells

59. Carcinogenesis Vascular dissemination and homing of tumor cells: Vascular dissemination and homing of tumor cells: May form emboli May form emboli Most travel as single cells Most travel as single cells Adhesion to vascular endothelium Adhesion to vascular endothelium extravasation extravasation

60. Carcinogenesis Main changes in the cell physiology that lead to formation of the malignant phenotype: Main changes in the cell physiology that lead to formation of the malignant phenotype: A- Self-sufficiency in growth signals B- Insensitivity to growth-inhibitory signals C- Evasion of apoptosis D- Limitless replicative potential E- Sustained angiogenesis F- Ability to invade and metastsize

61. Genomic Instability Enabler of malignancy Enabler of malignancy Due to defect in DNA repair genes Due to defect in DNA repair genes Examples: Examples: Hereditary Nonpolyposis colon carcinoma(HNPCC) Hereditary Nonpolyposis colon carcinoma(HNPCC) Xeroderma pigmentosum Xeroderma pigmentosum Familial breast cancer Familial breast cancer

62. Genomic Instability Familial breast cancer: Familial breast cancer: Due to mutations in BRCA1 and BRCA2 genes Due to mutations in BRCA1 and BRCA2 genes These genes regulate DNA repair These genes regulate DNA repair Account for 80% of familial breast cancer Account for 80% of familial breast cancer They are also involved in other malignancies They are also involved in other malignancies

63. Molecular Basis of multistep Carcinogenesis Cancer results from accumulation of multiple mutations Cancer results from accumulation of multiple mutations All cancers have multiple genetic alterations, involving activation of several oncogenes and loss of two or more tumor suppressor genes All cancers have multiple genetic alterations, involving activation of several oncogenes and loss of two or more tumor suppressor genes

64. Molecular Basis of multistep Carcinogenesis

65. Tumor progression Many tumors become more aggressive and acquire greater malignant potential…this is called “ tumor progression” …not increase in size!! Many tumors become more aggressive and acquire greater malignant potential…this is called “ tumor progression” …not increase in size!! By the time, the tumor become clinically evident, their constituent cells are extremely heterogeneous By the time, the tumor become clinically evident, their constituent cells are extremely heterogeneous

67. Karyotypic Changes in Tumors Translocations: Translocations: In CML : t(9,22) …” Philadelphia chromosome” In CML : t(9,22) …” Philadelphia chromosome” In Burkitt Lymphoma : t(8,14) In Burkitt Lymphoma : t(8,14) In Follicular Lymphoma : t(14,18) In Follicular Lymphoma : t(14,18) Deletions Deletions Gene amplification: Gene amplification: Breast cancer : HER-2 Breast cancer : HER-2

68. TranslocationsGene amplification